The nature of the protein-protein relationships which occur between membrane proteins and between membrane proteins and cytoplasmic proteins are not well understood. Also, it is not clear whether or not the alterations in the functions of the plasma membrane which occur during neoplastic transformation are related to changes in the supramolecular structure of the membrane. In this laboratory we have been developing methods to analyze protein-protein interactions using chemical cross-linking reagents such as DSP [dithiobis(succinimidylpropionate)]. We have found that we can cross-link the transferrin receptor HeLa cells into a Triton insoluble residue. Analyses on two-dimensional electrophoresis SDS-PAG (sodium dodecylsolfate polyacrylamide (nonreducing-reducing) gel systems reveal that a selective subset of proteins is lost from the Triton-soluble material released from 35S-methionine-labeled HeLa cells. Subsequent studies have shown that most of thecross-linked protein from HeLa cells treated with DSP does not enter SDS-PAG. To circumvent this, we have developed a two-dimensional gel system where the first dimension is an SDS-2% agarose gel (on Gel-Bond film) and the second dimension is an SDS-polyacrylamide gradient gel. Using this system, we have been able to resolve a large number of cross-linked protein complexes in the Triton insoluble matrix after it has been solubilized with hot 1% SDS. We are presently working on methods for biotinylating probe ligands so that we can isolate specific receptor complexes such as those containing the transferrin receptor and analyze the protein components of these complexes. These studies should have general applicability in deciphering the structure of membrane receptors and possible alteration in structure which occurs during growth, neoplastic transformation, or upon interaction of receptors with specific ligands. (A)